A replication-competent Ad4-HIVenv recombinant virus has been generated and is being characterized prior to production under appropriate conditions for use in a phase I clinical trial. An additional Ad4-HIVgag recombinant is also under development. For clinical grade production the candidate vaccine virus will be produced in human diploid cells. Exploration of the optimal method for virus production on these cells is on-going. Ad recombinants containing the green fluorescent protein (GFP) have been generated and are being used in studies in vitro for optimization of transfection procedures. In addition, use of these GFP recombinants in vivo has allowed biodistribution studies of the Ad-recombinant in the rhesus macaque model following administration by several different mucosal routes in order to determine which one(s) results in Ad-replication at gastrointestinal and genital/rectal sites. These sites are principal targets of HIV infection, and it is believed desirable that the vaccine virus replicate at these sites in order to elicit local mucosal immunity. Persistence of the Ad-recombinant virus will also be explored in vivo. New Ad-recombinants are under development both for future clinical use and for pre-clinical studies in the rhesus macaque model. These include recombinants containing novel envelope inserts intended to generate broad, potent neutralizing antibodies. This work includes assessment of immunogenicity of the newly constructed recombinant vaccines in appropriate animal models. Overall, this project is focused on moving the replication-competent Ad-recombinant vaccine approach into phase I clinical trials. This is a novel approach, which has shown superior immunogenicity in non-human primate studies in comparison to non-replicating Ad-recombinant vaccines.